CN114957434A

CN114957434A - Polypeptides for the treatment of autoimmune diseases

Info

Publication number: CN114957434A
Application number: CN202110205336.1A
Authority: CN
Inventors: 刘二龙; 胡柳益; 胡俊
Original assignee: Foshan Rexiu Biotechnology Co ltd
Current assignee: Foshan Rexiu Biotechnology Co ltd
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2022-08-30
Anticipated expiration: 2041-02-24
Also published as: CN114957434B; WO2022179093A1

Abstract

The present invention relates to the field of disease treatment. In particular, the invention provides polypeptides or variants thereof for use in the treatment of autoimmune diseases (e.g., systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis), fusion proteins comprising such polypeptides or variants thereof, and medical uses of such polypeptides or variants and fusion proteins thereof. The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a variant or fusion protein thereof, which can be used to treat an autoimmune disease (e.g., systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis) or alleviate one or more symptoms of an autoimmune disease.

Description

Polypeptides for the treatment of autoimmune diseases

Technical Field

The present invention relates to the field of disease treatment. In particular, the invention provides polypeptides or variants thereof for use in the treatment of autoimmune diseases (e.g., systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis), fusion proteins comprising such polypeptides or variants thereof, and medical uses of such polypeptides or variants and fusion proteins thereof. The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention, or a variant or fusion protein thereof, useful for treating an autoimmune disease (e.g., systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis), or alleviating one or more symptoms of an autoimmune disease.

Background

Autoimmune diseases are a series of diseases that cause organ and tissue damage due to impaired or lost self-tolerance of the immune system. Including, for example, Systemic Lupus Erythematosus (SLE), type 1 diabetes, Rheumatoid Arthritis (RA), and the like. Patients with autoimmune diseases have abnormally activated immune functions, attack normal tissues and organs of the body, and can involve multiple systemic diseases.

Systemic Lupus Erythematosus (SLE) is the name given for the English system lupus erythematosus. Systemic lupus erythematosus is an autoimmune disease, and occurs in women of 20-40 years old. The etiology of the disease is not clear up to now, and a large number of researches show that genetics, endocrine, infection, immune abnormality and some environmental factors are possibly related to the pathogenesis of the disease. Under the interaction of various factors such as genetic factors, environmental factors, estrogen level and the like, T lymphocytes of a patient are reduced, the function of inhibitory T cells is reduced, B cells are excessively proliferated, a large amount of autoantibodies are generated and combined with corresponding autoantigens in the body to form corresponding immune complexes which are deposited on the skin, joints, small blood vessels, glomeruli and other parts to cause acute and chronic inflammation and tissue necrosis (such as lupus nephritis), or the autoantibodies directly react with histocyte antigens to cause cell damage (such as the combination of specific antigens of erythrocytes, lymphocytes and blood platelet walls with corresponding autoantibodies to respectively cause hemolytic anemia, lymphopenia and thrombocytopenia), so that the body is damaged by multiple systems.

Diagnosis of SLE relies primarily on clinical manifestations, laboratory examinations, histopathology and imaging examinations. In the classification criteria for SLE revised by the American College of Rheumatology (ACR) in 1997, laboratory tests such as hematological abnormalities, immunological abnormalities and autoantibody positivity are clearly included as diagnostic criteria. Because systemic lupus erythematosus patients often have blood system abnormality, kidney damage and the like, the routine blood examination can have symptoms of anemia, reduction of white blood cell count, reduction of platelets and the like; urinalysis can reveal proteinuria, hematuria, cell and granular casts. Currently, the conventional detection items of the systemic lupus erythematosus-associated autoantibodies clinically developed mainly include antinuclear antibodies (ANA), anti-double-strand deoxyribonucleic acid antibodies (anti-dsDNA antibodies), anti-soluble antigen antibodies (anti-ENA antibodies) (including anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-rRNP, anti-Scl-70, anti-Jo-1 and the like), anti-nucleosome antibodies, anti-phospholipid antibodies and the like. In the ACR revised SLE classification criteria, immunological abnormalities and autoantibody positivity include: anti-Sm antibody, anti-dsDNA antibody, anti-phospholipid antibody and ANA positive.

Type 1 diabetes mellitus, type 1 diabetes mellitis, abbreviated as T1 DM. T1DM is an autoimmune disease caused by destruction of the beta cells in the islets of langerhans by the immune system, and T1DM can occur at any age, but is most commonly seen in and begins to attack around puberty. Studies have shown that both genetic and environmental factors play a role in the pathogenesis of T1DM, but the exact cause of T1DM is not clear. The current major treatment modality for T1MD remains lifelong insulin injection for glycemic control.

Rheumatoid arthritis, abbreviated as RA. RA is characterized by an imbalance in the immune system that leads to overproduction of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF) interleukin 1(IL-1) and a deficiency of anti-inflammatory cytokines such as IL-10, IL-11. RA is characterized by synovial inflammation, which progresses to cartilage destruction, bone erosion and subsequent joint deformity. The primary symptoms of RA are joint inflammation, swelling, difficulty moving and pain. In the late stages of RA, enzymes produced by the inflammatory cells digest bone and cartilage. Long-term injury leads to chronic pain, loss of function, deformity and even a shortened lifespan of the joint. Although anti-TNF therapy has made good progress in the treatment of RA in recent years, it is only available to a subset of patients, at least one third of whom do not respond. Thus, successful treatment of arthritis remains an unresolved medical need.

The autoimmune diseases have strong heterogeneity, most patients are not cured radically no matter the traditional Chinese medicine or western medicine is adopted for treatment, the disease condition can be relieved only through medicines, obvious toxic and side effects can be brought after long-term administration, and huge economic and mental burdens are brought to the patients and families, so that the development of a new scheme and a new medicine for treating the autoimmune diseases have important social and economic significance.

Disclosure of Invention

Heat Shock Proteins (HSPs) are a class of proteins that are highly conserved in biological evolution and widely found in prokaryotes and eukaryotes. HSP can be divided into multiple subfamilies such as HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSP and ubiquitin according to the homology degree and molecular weight. The human heat shock protein 90 family (HSP90) includes four members of HSP90 alpha, HSP90 beta, gp96(grp94) and Trap-1. gp96(GRP94) is representative of the HSP90 family of endoplasmic reticulum, is highly homologous to cytoplasmic HSP90, and has the following major biological functions: molecular chaperones, which participate in the folding and assembly of newly synthesized proteins; binding to other peptidic proteins in the cell, especially denatured proteins, and participating in processes of cellular resistance to damage, repair, and heat tolerance; participating in the process of proteolysis; combining with antigen peptide, processing and presenting tumor antigen and maintaining intracellular environment; has certain regulation effect on the growth, development, differentiation and death of cells.

It has been reported that full-length gp96 can activate regulatory T cells (Tregs), but it also has a strong activating effect CD4 ⁺ T、CD8 ⁺ The functions of T cells, B cells and the like cannot specifically and independently target regulatory T cells, so that the full-length gp96 has the possibility of activating the immune system in the treatment of autoimmune diseases, resulting in poor treatment effect and even aggravating the autoimmune diseases.

The inventors of the present application have unexpectedly found, after a great deal of research, that a specific polypeptide fragment of full-length gp96 has activities of specifically and individually targeting regulatory T cells and not targeting effector T cells and B cells, and thus is particularly suitable for the treatment of autoimmune diseases, and has important clinical application values.

Isolated polypeptide or variant thereof

In one aspect, the invention provides an isolated polypeptide or variant thereof, wherein the polypeptide consists of at least 136 contiguous amino acid residues of gp96 protein and comprises: amino acid residues 578-713 of the gp96 protein;

wherein the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1 or a few (e.g., 1, 2, 3, 4 or 5) amino acid residues and retains the biological function of the polypeptide from which it is derived.

In this context, the biological functions of the polypeptide of the invention or a variant thereof include, but are not limited to, inducing regulatory T cell activation, inducing no or substantially no effector T cell and B cell activation, treating autoimmune diseases, reducing the level of anti-double stranded DNA antibodies, reducing the level of urine protein, and/or reducing blood glucose.

It is known to those skilled in the art that during translation of mRNA, the first amino acid (e.g., methionine (M)) encoded by the start codon is often found in the polypeptide chain produced as a result of the action of the start codon. Thus, the polypeptide of the present invention or a variant thereof encompasses not only an amino acid sequence not containing an amino acid (e.g., methionine) encoded by the initiation codon at its N-terminus but also an amino acid sequence containing an amino acid (e.g., methionine) encoded by the initiation codon at its N-terminus.

In certain embodiments, the gp96 protein is of human origin. In certain embodiments, the gp96 protein has an amino acid sequence as set forth in SEQ ID NO 7.

In certain embodiments, an isolated polypeptide of the invention consists of no more than 254 contiguous amino acid residues of gp96 protein, e.g., no more than 250, 240, 230, 226, 224, 220, 210, 200, 196, 194, 190, 180, 170, 166, 164, 160, 150, 140, or 136 contiguous amino acid residues.

In certain embodiments, the variant differs from the polypeptide from which it is derived only in the substitution, deletion, or addition of 1 or a few (e.g., 1, 2, 3, 4, or 5) amino acid residues, which substitution is a conservative substitution.

In certain embodiments, the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1, 2 or 3 amino acid residues. In certain embodiments, the substitution comprises replacing the amino acid residue with alanine (a).

In certain embodiments, the polypeptides of the invention comprise: the protein gp96 comprises the amino acid residues at 578-position 713, 578-position 743, 578-position 773, 578-position 803, 550-position 713, 550-position 743, 550-position 773 or 550-position 803.

In certain embodiments, the isolated polypeptide comprises, or consists of, an amino acid sequence selected from the group consisting of: 30, 31, 32, 33, 37, 42, 43, 44 SEQ ID NOs. The sequence shown here contains a methionine at its N-terminus encoded by the start codon. It will be appreciated by those skilled in the art that the isolated polypeptide may also comprise or consist of the amino acid sequence described above which does not comprise the methionine encoded by the start codon at its N-terminus.

In certain embodiments, the variant comprises, or consists of, an amino acid sequence selected from the group consisting of: SEQ ID NOs: 55-62. The sequence shown here contains a methionine at its N-terminus encoded by the start codon. It is understood by the person skilled in the art that the variant may also comprise or consist of the above-mentioned amino acid sequence not comprising the methionine encoded by the start codon at its N-terminus.

Fusion proteins

In another aspect, the invention provides a fusion protein comprising an isolated polypeptide of the invention (or variant thereof) and an additional polypeptide.

In certain embodiments, the additional polypeptide is selected from a protein tag, a targeting moiety, or any combination thereof.

Herein, protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and the skilled artisan knows how to select an appropriate protein tag according to the desired purpose (e.g., purification, detection, or tracking).

The term "targeting moiety" as used herein refers to a domain capable of directing a polypeptide of the invention (or a variant thereof) to a desired location, which may be a specific tissue, a specific cell, or even a specific intracellular location (e.g., nucleus, ribosome, endoplasmic reticulum, lysosome, or peroxisome). The skilled person knows how to design the corresponding targeting domain by the properties of the desired position. In certain embodiments, the targeting moiety comprises a ligand, receptor, or antibody or binding domain thereof.

In certain embodiments, the additional polypeptide is linked to the N-terminus or C-terminus of the polypeptide of the invention (or variant thereof), optionally via a linker. In certain embodiments, the linker is a sequence comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Gly or Ser).

Preparation of polypeptides and fusion proteins

The polypeptide of the present invention or a variant or fusion protein thereof is not limited by the manner of production thereof, and may be produced, for example, by genetic engineering methods (recombinant techniques) or by chemical synthesis methods.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the invention, or a variant or fusion protein thereof.

In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (i) a sequence shown as any one of SEQ ID NOs 13-16, 20, 25-27 and 47-54; (ii) (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity compared to the sequence of (i); (iii) (iii) a sequence that hybridizes under stringent conditions to the sequence described in (i) or (ii); or (iv) the complement of the sequence described in (i) or (ii).

In another aspect, the present invention also provides a vector comprising an isolated nucleic acid molecule as described above. The vector of the present invention may be a cloning vector or an expression vector. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages, cosmids, and the like.

In another aspect, the invention also provides a host cell comprising the isolated nucleic acid molecule or vector of the invention. Such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells (e.g., Sf9 cells), plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

In another aspect, the invention also provides a method of producing a polypeptide of the invention or a variant or fusion protein thereof, comprising culturing a host cell of the invention under conditions which allow expression of the polypeptide or variant or fusion protein thereof, and recovering the polypeptide or variant or fusion protein thereof from the cultured host cell culture.

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising an isolated polypeptide (or variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition comprises one or more of the isolated polypeptide (or variant thereof) or fusion protein of the invention.

The polypeptide (or a variant thereof), the fusion protein or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, lyophilized powders) and the like. In some embodiments, the polypeptide (or variant thereof), fusion protein, or pharmaceutical composition of the invention may be formulated as an injection solution or a lyophilized powder.

In addition, the polypeptide (or variant thereof) or fusion protein of the invention may be present in a pharmaceutical composition in unit dosage form for ease of administration.

The polypeptide (or variant thereof), fusion protein, or pharmaceutical composition of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention is administered by intravenous infusion or injection.

The polypeptides (or variants thereof), fusion proteins or pharmaceutical compositions provided herein can be used alone or in combination, or in combination with additional pharmaceutically active agents (e.g., anti-inflammatory drugs or immunosuppressive agents). Such additional pharmaceutically active agents may be administered prior to, concurrently with, or subsequent to the administration of the polypeptide (or variant thereof), fusion protein, or pharmaceutical composition of the invention.

In certain embodiments, the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent, such as a drug having activity in treating autoimmune disease. In certain embodiments, the additional pharmaceutically active agent is selected from an anti-inflammatory drug or an immunosuppressive agent, such as a non-steroidal anti-inflammatory drug, a steroidal anti-inflammatory drug, an antibody or antagonist to an inflammatory cytokine, an anti-inflammatory cytokine, and the like.

Therapeutic uses

The inventors of the present application have for the first time found that the C-terminal domain of gp96 or an active fragment thereof is capable of specifically activating regulatory T cells without activating the activity of effector T cells and B cells, and is thus particularly suitable for the treatment of autoimmune diseases.

Thus, in another aspect, the present invention also relates to the use of the C-terminal domain of gp96 or an active fragment or variant thereof, or the isolated polypeptide of the invention (or variant thereof), the fusion protein, the isolated nucleic acid molecule, the vector or the host cell of the invention in the preparation of a medicament for preventing and/or treating an autoimmune disease, or reducing the level of anti-double stranded DNA antibodies, or reducing the level of urine protein, or reducing blood glucose in a subject.

In certain embodiments, the C-terminal domain consists of amino acid residues 550-803 of the gp96 protein. In certain embodiments, the C-terminal domain has the sequence shown as SEQ ID NO 30.

In certain embodiments, the active fragment of the C-terminal domain of gp96 or variant thereof is selected from the group consisting of an isolated polypeptide of the invention or variant thereof.

In certain embodiments, the agent comprises one or more of the C-terminal domain of gp96 or an active fragment or variant thereof, an isolated polypeptide (or variant thereof) of the invention, or a fusion protein.

In certain embodiments, the autoimmune disease may be selected from systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, crohn's disease, myasthenia gravis, or polymyositis.

In certain embodiments, the medicament is for preventing and/or treating systemic lupus erythematosus or ameliorating one or more symptoms of systemic lupus erythematosus (e.g., reducing the level of anti-double stranded DNA antibodies, and/or reducing the level of urine proteins).

In certain embodiments, the medicament is for preventing and/or treating type 1 diabetes or ameliorating one or more symptoms of type 1 diabetes (e.g., lowering blood glucose).

In certain embodiments, the medicament is for preventing and/or treating rheumatoid arthritis or alleviating one or more symptoms of rheumatoid arthritis (e.g., reducing joint swelling, tenderness, and/or pain).

In certain embodiments, the subject may be a mammal, such as a human or a mouse. In certain embodiments, the subject has, or is suspected of having, or is at risk of having, an autoimmune disease. In certain embodiments, the subject has, or is suspected of having, or is at risk of having, systemic lupus erythematosus. In certain embodiments, the subject has, or is suspected of having, or is at risk of having, type 1 diabetes. In certain embodiments, the subject has, or is suspected of having, or is at risk of having, rheumatoid arthritis.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, virological, biochemical, immunological laboratory procedures used herein are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "gp 96," also known as Grp94, is a member of the heat shock protein 90 family located on the endoplasmic reticulum membrane of cells. The gp96 protein consists of an N-terminal domain (N-terminal ATP-binding domain), an M-domain (charged middle domain), and a C-terminal domain (C-terminal homodimeric domain). gp96 is well known to those skilled in the art, and its sequence can be found in various public databases, such as NCBI GENBANK database accession numbers: AAH 66656.1.

As used herein, when referring to the amino acid sequence of gp96 protein, it uses the amino acid sequence of SEQ ID NO:7, to be described. For example, the expression "amino acid residues 578-713 of the gp96 protein" means that the amino acid sequence shown in SEQ ID NO:7 at amino acid residue 578-713. However, it is understood by those skilled in the art that mutations or variations can be naturally occurring or artificially introduced in the amino acid sequence of gp96 without affecting its biological function. Thus, in the present invention, the term "gp 96" and its analogous expressions shall include all such sequences, including, for example, SEQ ID NO:7 and natural or artificial variants thereof. Also, when describing a sequence fragment of gp96 protein, it includes not only the sequence of SEQ ID NO:7, and also includes the corresponding sequence fragments in natural or artificial variants thereof. For example, the expression "amino acid residues 578-713 of the gp96 protein" includes the amino acid sequence of SEQ ID NO:7 at amino acid residue 578-713 and the corresponding fragment in a variant (natural or artificial) thereof. According to the invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragments at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

As used herein, "stringent conditions" for hybridization refer to conditions under which specific hybrids are formed but non-specific hybrids are not formed. Typical stringent conditions include conditions for hybridization at a potassium concentration of about 25mM to about 50mM and a magnesium concentration of about 1.0mM to about 5.0 mM. MakingFor example, stringent conditions may refer to conditions in Tris-HCl (pH8.6), 25mM KCl, and 1.5mM MgCl ₂ Conditions for hybridization are not limited thereto. Those skilled in the art can easily select such conditions by changing the hybridization reaction, the salt concentration of the hybridization reaction solution, and the like.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithms of e.meyers and w.miller (comput.appl biosci., 4:11-17(1988)) which have been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table (weight residue table), a gap length penalty of 12 and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA 94:412-417(1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R.Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)) which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans. In certain embodiments, the subject (e.g., human) has an autoimmune disease.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and includes, but is not limited to: pH adjusting agents, surfactants, ionic strength enhancers, agents to maintain osmotic pressure, agents to delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament (e.g., inhibitory activity against PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "treating" refers to treating or curing a disease (e.g., an autoimmune disease), delaying the onset of one or more symptoms of a disease, and/or delaying the progression of a disease.

As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a therapeutically effective amount can be an amount effective or sufficient to treat or cure a disease (e.g., an autoimmune disease), delay the onset of one or more symptoms of a disease, and/or delay the progression of a disease. Such an effective amount can be readily determined by one of skill in the art or a physician, and can be related to the intended purpose, the general health of the subject, the age, sex, weight, severity of the disease to be treated, complications, mode of administration, and the like. Determination of such effective amounts is well within the capability of those skilled in the art.

As used herein, the biological function of a polypeptide of the invention or a variant thereof includes, but is not limited to, one or more selected from the group consisting of:

1) inducing regulatory T cell activation;

2) does not induce effector T cell and B cell activation;

3) treating an autoimmune disease (e.g., systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis) in a subject;

4) reducing the level of anti-double stranded DNA antibodies in the subject;

5) reducing the level of urinary protein in the subject;

6) lowering blood glucose in a subject.

Advantageous effects

The polypeptides of the invention (or variants thereof) and fusion proteins comprising the polypeptides (or variants thereof) have significant advantages over the prior art. In particular, the polypeptides (or variants thereof) and fusion proteins of the invention specifically induce regulatory T cell activation and do not induce effector T cell and B cell activation, thereby avoiding the potential risk that full-length gp96 protein may exacerbate the progression of autoimmune disease. In addition, the ability of the polypeptide (or the variant thereof) and the fusion protein of the invention to induce the activation of regulatory T cells is obviously better than that of full-length gp96 protein, and the polypeptide (or the variant thereof) and the fusion protein have better immunoregulatory activity. Therefore, the polypeptide (or variant thereof) and the fusion protein of the invention are particularly suitable for the treatment of autoimmune diseases, and have great clinical value.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows the percentage of regulatory T cells (Tregs) (% CD 3) in mice immunized with 100. mu.g of different gp96 protein fragments ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs/CD3 ⁺ CD4 ⁺ T cells). A, P<0.05；**,P<0.01

FIG. 2 shows the percentage of regulatory T cells (Tregs) (% CD 3) in mice immunized with 300. mu.g of different gp96 protein fragments ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs/CD3 ⁺ CD4 ⁺ T cells). A, P<0.05；***,P<0.001

FIG. 3 shows the percentage of regulatory T cells (Tregs) (% CD 3) in mice immunized with 500. mu.g of different gp96 protein fragments ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs/CD3 ⁺ CD4 ⁺ T cells). A, P<0.05；***,P<0.001

FIG. 4 shows the percentage of regulatory T cells (Tregs) in mice after immunization of mice with 300. mu.g of different polypeptide fragments and full-length gp96 (% CD 3) ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs/CD3 ⁺ CD4 ⁺ T cells). Peptides compared to gp96 group P <0.01

FIG. 5 shows CD4 activated after immunization of mice with 30. mu.g or 300. mu.g of different polypeptide fragments and full-length gp96 in combination with ovalbumin (NP-OVA) ⁺ Percentage of T cells (INF. gamma.) ⁺ CD4 ⁺ T/CD4 ⁺ T)。**，P<0.01；***,P<0.001

FIG. 6 shows CD8 activated after immunization of mice with 30. mu.g or 300. mu.g of different polypeptide fragments and full-length gp96 in combination with ovalbumin (NP-OVA) ⁺ Percentage of T cells (INF. gamma.) ⁺ CD8 ⁺ T/CD8 ⁺ T)。**，P<0.01；***,P<0.001

FIG. 7 is the level of anti-OVA antibody IgG in the serum of mice immunized with 300. mu.g of different polypeptide fragments and full-length gp 96. P <0.01

FIG. 8 shows the level of anti-dsDNA antibodies in the serum of mice immunized with Lyn (-/-) mice with 100. mu.g, 300. mu.g or 500. mu.g of different polypeptide fragments and full-length gp 96. At the same dose, P <0.001 (") in the gp96 group compared to the PBS group, P <0.05 (") in the peptide group compared to the gp96 group

FIG. 9 shows the levels of protein in the urine of mice immunized with Lyn (-/-) mice with 100. mu.g, 300. mu.g or 500. mu.g of different polypeptide fragments and full-length gp 96. At the same dose, P <0.001 (") in the gp96 group compared to the PBS group, P <0.05 (") in the peptide group compared to the gp96 group

FIG. 10 shows the prevalence of NOD mice in the prophylactic model T1D after immunization with 100. mu.g, 300. mu.g, or 500. mu.g of different polypeptide fragments and full-length gp 96. At the same dose, the peptide groups compared with gp96 groups were either P <0.05 (") or P <0.01 (". ")

FIG. 11 shows the blood glucose levels in the blood of NOD mice in prophylactic models immunized with 100. mu.g, 300. mu.g, or 500. mu.g of different polypeptide fragments and full-length gp 96. At the same dose, P <0.05 (") in peptide versus gp96 groups

FIG. 12 is a graph of activated CD8 in spleen cells of NOD mice protected from full length gp96 after immunization with 100. mu.g, 300. mu.g, or 500. mu.g of different polypeptide fragments ⁺ Percentage of T cells (INF. gamma.) ⁺ CD8 ⁺ T/CD8 ⁺ T). At the same dose, P <0.05 (") in peptide versus gp96 groups

FIG. 13 is the blood glucose levels in blood of NOD mouse treatment models immunized against 300 μ g of different polypeptide fragments and full length gp 96. At the same dose, P <0.05 (") in peptide versus gp96 groups

FIG. 14 is the blood glucose levels in the blood of NOD mouse treatment models immunized with 300 μ g Peptide1 and its amino acid deletions and substitution mutations.

FIG. 15 is the blood glucose levels in the blood of NOD mouse treatment models immunized with 300 μ g of Peptide15 and its amino acid deletions and substitution mutations.

FIG. 16 shows the results of detecting rheumatoid inflammation index in the induced mouse joint inflammation model after different polypeptide fragments and full-length gp96 immunization.

Sequence information

Table 1: information on the sequences to which the present application relates is described in the following table.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. The experimental procedures in the examples are conventional unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the quantitative experiments in the examples, three repeated experiments are set, and the results are averaged.

The sources of experimental materials referred to in the following examples are as follows:

the female Lyn (-/-) knockout murine systemic lupus erythematosus model is described in: hibbs ML, et al, multiple defects in the animal system of Lyn-specific mice, cutting in automatic animal disease. cell (1995)83(2): 301-11; yu CC, Yen TS, Lowell CA, DeFranco AL. Lupus-like kidney disease in micro discovery in the Src family kinase Lyn and Fyn. Current biol.2001 Jan 9; 11(1):34-8.

Female NOD/LTJ mice are described in the following references: terry L.et al.I The Nonobese Diabetes mellitus as a Model of Autoimmune Diabetes, Immune Dysregelation Gets The NOD.Immunity, December,1997, Vol.7, 727-738; michelle Solomon, Nora Sarvetnick, et al, the Pathologenetics of Diabetes in the NOD mouse, immunology,2004, 84:239-64.

Sf9 cells were purchased from Invitrogen, catalog No.: 11496-;

pFastBac

^TM 1 plasmid purchased from Invitrogen, catalog No.: 10359-016；

DH10Bac ^TM Competent cells were purchased from Invitrogen, catalog No.: 10361-012;

Insect-XPRESSTM Protein-free Instrument with L-Glutamine from LONZA, catalog No.: 12-730Q;

ultrafiltration tubes were purchased from Merck Millipore, catalog No.: UFC 905096;

ELISA kits were purchased from eBioscience, catalog No.: BMS614 INST;

ni affinity chromatography pre-column purchased from alatin, catalog No.: n5289-01;

superdex 20010/300 GL molecular sieve columns from GE, Catalogue No.: 17517501, respectively;

coli DH10Bac competent cells were purchased from bekka biotechnology limited, beijing original, catalog No.: CL 108-01.

Example 1: preparation of full-Length gp96 or different gp96 truncates

1. And (5) constructing a recombinant plasmid.

Recombinant plasmid pFastBac1-DNAs containing the coding sequence of full-length gp96 or different gp96 truncations were synthesized by King-Share Biotechnology GmbH. The amino acid sequences and coding nucleic acid sequences of the full-length gp96 or the various gp96 truncations involved are shown in Table 1. The recombinant plasmids were transformed into DH10Bac, respectively ^TM And (4) carrying out recombination screening on the competent cells to obtain recombinant bacmid DNA. All gp96 protein fragments and polypeptides were C-terminally tagged with 6XHis, the DNA sequence of the tag being CACCACCACCATCACCAC (SEQ ID NO: 63).

2. Expression of full-length gp96 or a different gp96 truncation.

2.1 Co-transfection of recombinant bacmid DNA into adherent Sf9 cells (per 8X 10) ⁵ About 2. mu.g of recombinant plasmid was transfected into each Sf9 cell; in the co-transfection process, the transfection reagent was Cellfectin II reagent (purchased from Life technologies, Cat. No.: 10362-.

2.2 Sf9 cell suspension 1 (containing 8X 10 cells) ⁶ Sf9 cells) at 27 ℃ for 1-5 h to obtain adherent culture cells(ii) a And then adding P1 virus generations (with the dose of 0.05-0.1 MOI) into the adherent culture cells, incubating for 72h at 27 ℃, and centrifuging for 5min at 4000rpm to obtain supernatant, namely the P2 virus.

2.3 Sf9 cell suspension 2 (containing 8X 10 cells) ⁶ Sf9 cells) are added with P2 generation virus (the dosage is 0.05-0.1 MOI), cultured for 72h at the temperature of 27 ℃ and at the rpm of 100-120, and centrifuged for 5min at the rpm of 4000 for obtaining the supernatant which is the P3 generation virus.

3. Purification of full-length gp96 or different gp96 truncations.

3.1 to 300ml of Sf9 cell suspension 3 (containing Sf9 cells 2-4X 10) ⁶ Adding P3 generation virus (the dose is 0.05MOI) into the suspension, and culturing at the temperature of 27 ℃ and the rpm of 100-120 for 72-96 h to obtain suspension.

3.2 taking the suspension, 7000rpm centrifugation for 20min, obtain supernatant 1.

3.3 filtering the supernatant fluid 1 by a filter membrane of 0.22mm to obtain a supernatant fluid.

3.4 loading the sample loading solution on a Ni affinity chromatography column. The flow rate of the sample is controlled at 1ml/min, and then the sample is washed with an imidazole-free Tris-HCl buffer solution, eluted with an imidazole-containing Tris-HCl buffer solution, and the eluate is harvested. Ultrafiltering the eluate with ultrafiltration tube with cut-off molecular weight of 10KD, and concentrating to obtain concentrated solution of about 1 ml. The concentrated solution contains recombinant gp 96C-terminal protein.

3.5 the concentrate obtained in step 4 was applied to Superdex 20010/300 GL molecular sieve chromatography column (flow rate 0.25ml/min), washed with PBS buffer containing 150mM NaCl (flow rate 0.25ml/min) at pH7.5, and the permeate was collected as the corresponding protein peak, and the corresponding protein was detected by SDS-PAGE, and the purified protein was further identified by western blotting using anti-His-tag antibody (purchased from Shanghai Biyuntian Biotech Co., Ltd.). And further carrying out ultrafiltration concentration by adopting an ultrafiltration tube with the molecular weight cutoff of 10KD to obtain solutions respectively containing different gp96 protein fragments and polypeptides. And (3) determining the protein concentration in the solution of the protein and the polypeptide by using a BCA method, finally subpackaging, wherein the protein concentration is 1mg/ml, and storing at-80 ℃.

Example 2: evaluation of the Activity of full-Length gp96 or different gp96 truncations induced regulatory T cells in mice

This example examines the conditions of the full-length gp96 protein (SEQ ID NO:7), the truncated gp96-N (SEQ ID NO:8), gp96-M (SEQ ID NO:10), gp96-N + M (SEQ ID NO:9), gp96-C (SEQ ID NO:11) and gp96-C + M (SEQ ID NO:12) inducing mice to produce regulatory T cells. The C-terminal of each protein or polypeptide is added with a 6xHis tag. Wherein gp96-N corresponds to amino acid residue 550-578 of the full-length protein; gp96-M corresponds to amino acid residue 578-713 of the full-length protein; gp96-C corresponds to amino acid residues 713-803 of the full-length protein.

1. Group immunization of mice

105 six-week-old C57BL/6 mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd.) weighing 14-16 g were randomly divided into a gp96 treatment group, a gp96-N treatment group, a gp96-M treatment group, a recombinant gp96-N + M treatment group, a gp96-C treatment group, a gp96-C + M treatment group and a control group (5 mice per group), and the following treatments were performed, respectively:

gp96-N treatment group: mice were grown to age 9 weeks and a solution of purified gp96-N prepared according to example 1 was injected subcutaneously into the abdomen and scored as day 1 of the experiment; after the 8 th day of the experiment, a solution of purified gp 96N obtained from the preparation of example 1 was re-injected ventrally subcutaneously; on day 22 of the experiment, a solution of purified gp96-N obtained from the preparation of example 1 was re-injected subcutaneously ventrally. Each injection was given at 100. mu.g, 300. mu.g or 500. mu.g gp96-N per mouse.

gp96-M treatment group: mice were grown to 9 weeks of age and a solution of purified gp96-M prepared according to example 1 was injected subcutaneously into the abdomen and scored as day 1 of the experiment; after the 8 th day of the experiment, the solution of purified gp96-M obtained in example 1 was re-injected subcutaneously in the abdomen; on day 22 of the experiment, a solution of purified gp96-M obtained from the preparation of example 1 was re-injected subcutaneously ventrally. Each injection dose is 100 ug, 300 ug or 500 ug gp 96-M/injection.

gp96-N + M treatment group: mice grown to age 9 weeks and were injected ventrally subcutaneously with the purified gp96-N + M solution prepared in example 1, identified as experiment day 1; after the 8 th day of the experiment, purified gp96-N + M solution obtained from the preparation of example 1 was re-injected ventrally subcutaneously; on day 22 of the experiment, a solution of purified gp96-N + M prepared in example 1 was again injected subcutaneously in the abdomen. Each injection dose is 100 ug, 300 ug or 500 ug gp96-N + M/mouse.

gp96-C treatment group: mice were grown to age 9 weeks and were injected ventrally subcutaneously with purified gp96-C prepared in example 1, day 1 of the experiment; after the 8 th day of the experiment, a solution of purified gp96-C prepared in example 1 was re-injected ventrally subcutaneously; on day 22 of the experiment, a solution of purified gp96-C prepared in example 1 was re-injected subcutaneously ventrally. Each injection dose is 100 ug, 300 ug or 500 ug gp 96-C/injection.

gp96-C + M treatment group: mice grown to age 9 weeks, were injected ventrally subcutaneously with purified gp96-C + M prepared in example 1, as day 1 of the experiment; after the 8 th day of the experiment, a solution of purified gp96-C + M prepared in example 1 was re-injected ventrally subcutaneously; on day 22 of the experiment, a solution of purified gp 96C-3 prepared in example 1 was re-injected subcutaneously ventrally. Each injection dose is 100 μ g, 300 μ g or 500 μ g gp96-C + M/mouse.

Full length gp96 treatment group: mice grown to age 9 weeks, were injected ventrally subcutaneously with purified gp96-C + M prepared in example 1, as day 1 of the experiment; after the 8 th day of the experiment, a solution of purified gp96-C + M prepared in example 1 was re-injected ventrally subcutaneously; on day 22 of the experiment, a solution of full-length gp96 prepared in example 1 was re-injected ventrally subcutaneously. Each injection dose was 100. mu.g, 300. mu.g or 500. mu.g gp 96/mouse.

Control group: on the 1 st day of the experiment, the abdomen is injected with PBS buffer solution with pH value of 7.2 and 0.01mol/L, and on the 8 th day of the experiment, the abdomen is injected with PBS buffer solution with pH value of 7.2 and 0.01mol/L again; on day 22 of the experiment, another abdominal subcutaneous injection of 0.01mol/L PBS buffer, pH7.2 was performed. Each injection of PBS dose is 100 u l/body, 300 u g or 500 u l/body.

2. Evaluation of immune Effect

Mice were sacrificed on day 25, spleen was removed and mouse spleen lymphocytes were prepared and analyzed for the percentage of regulatory T cells (Tregs) (% CD 3) using flow cytometry ⁺ CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs/CD3 ⁺ CD4 ⁺ T cells).Methods for isolating and detecting regulatory T cells are described in Xinghui Li, et al.2013. introduction of regulatory T cells by high-dose gp96 supressures muscle promoter animal hyperactivation. PLoS one.8(7): e 68997.

The detection results are shown in FIGS. 1-3, and the percentages of regulatory T cells (Tregs) of mice after mice are immunized by different gp96 protein fragments of 100. mu.g, 300. mu.g and 500. mu.g respectively in FIGS. 1-3. The results show that the percentage of regulatory T cells of mice immunized with 100. mu.g, 300. mu.g or 500. mu.g of gp96-C, gp96-C + M protein is significantly higher than that of control group (PBS) or gp96-N, gp96-M, gp96-N + M immunized group (P <0.05 or P <0.001), and the levels of regulatory T cells of gp96-C and gp96-C + M protein immunized groups are significantly higher than that of full-length gp96 immunized groups (P <0.05), while the percentages of regulatory T cells of gp96-N, gp96-M, gp96-N + M protein and control group-treated mice are not significantly different.

Example 3: preparation of different truncations based on the C-terminal domain of gp96 protein and evaluation of the Activity thereof to induce regulatory T cells in mice

1. Design of different polypeptide fragments

The full-length C-terminal of gp96 has 255 amino acid residues, and is divided into 17 different polypeptide fragments: peptide 1-Peptide 17, the amino acid sequences of which are respectively shown in SEQ ID NOs: 30-46. The above 17 polypeptides were expressed and prepared by adding a 6xHis tag to the C-terminal of each of the above polypeptides by the method of example 1.

2. Group immunization of mice

95C 57BL/6 mice with the weight of 14-16 g at six weeks of age were randomly divided into 19 groups, and a Peptide 1-Peptide 17 experimental group and PBS and gp96 control groups were respectively arranged, and each group had 5 mice.

The mice grow to 9 weeks of age, and are injected with corresponding protein (polypeptide) or PBS subcutaneously in the abdomen on the 1 st day of the experiment, and are injected subcutaneously in the abdomen again on the 8 th day of the experiment; the experiment was performed again on day 22, abdominal subcutaneous injection. The dose of each injection is 300 mug/body.

3. Evaluation of Effect

The mice are sacrificed at the 25 th day, spleen of the mice is taken and separated to prepare spleen lymphocytes of the mice, and the flow cytometry is used for analyzing the percentage of the regulatory T cells of the mice after different recombinant polypeptide fragments are immunized. Methods for isolation and detection of regulatory T cells are as in example 2.

The detection results are shown in FIG. 4, and the results show that the percentage of the mouse regulatory cells in the Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 treatment groups is obviously higher than that in the control group (PBS) (P <0.001) and all the groups are higher than that in the full-length gp96 treatment group (P < 0.01). And it can be seen that the amino acid sequence contained in Peptide15 (amino acid residue 578-713 of the full-length gp96 protein) is an essential sequence for activating regulatory T cells.

Example 4: evaluation of mouse Effector T cell Activity induced by different truncations based on the C-terminal Domain of the gp96 protein

1. Group immunization of mice

110C 57BL/6 mice with the body weight of 14-16 g at six weeks of age were randomly divided into 22 groups, each of which was designated Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, Peptide15 polypeptide, full-length gp96 combined with ovalbumin (NP-OVA) (purchased from Beijing Boolpek Biotech Co., Ltd., product number: T-5051-100 x100 mg), individual NP-OVA or PBS control group (5 mice per group).

Mice grow to 9 weeks of age, and are injected with corresponding polypeptide or full-length gp96 combined with NP-OVA or NP-OVA alone or PBS for immunization in abdominal subcutaneous injection on the 1 st day of experiment, and are injected again in abdominal subcutaneous injection on the 8 th day of experiment; the experiment was performed again on day 22, abdominal subcutaneous injection. The dose of each injection of the polypeptide or the full-length gp96 is 30 mu g or 300 mu g per mouse, and the dose of the OVA protein is 20 mu g per mouse.

2. Evaluation of Effect

Mice were sacrificed on day 25, spleen cells of the mice were isolated, and the mice activated CD4 after immunization was analyzed by flow cytometry ⁺ T cell, CD8 ⁺ Percentage of T cells. Activated CD4 ⁺ T cell, CD8 ⁺ T cell separation and detection methods are described in Xinghui Li, et al.2013. indication of regulatory T cells by high-dose gp96 supress series muscle promoter animal hyper activity PLoS one.8(7): e 68997.

Activated CD4 ⁺ T cell, CD8 ⁺ The results of the T cell percentage measurements are shown in FIGS. 5 and 6, respectively, and show that mice in the groups treated with 30. mu.g and 300. mu.g of Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptide fragments at the immunizing doses were activated CD4 ⁺ T cells and CD8 ⁺ The percentage of T cells was not significantly different from the control group (PBS), whereas the 30 μ g immunization dose of full-length gp96 group mice activated CD4 ⁺ T cells and CD8 ⁺ The percentage of T cells was significantly higher than control (PBS) (P)<0.001) and OVA immunized group (P)<0.01), 300 μ g immunization dose of full-length gp96 group mice activated CD4 ⁺ T cells and CD8 ⁺ The percentage of T cells was significantly higher than control (PBS) (P)<0.01), which indicates that the polypeptide fragment does not activate effector T cells when immunized, and that full-length gp96 can obviously activate effector T cells.

The level of anti-OVA antibody IgG in the serum of mice immunized with 300. mu.g of different polypeptide fragments and full-length gp96 was further determined by ELISA, as described in detail in Bettina Eide Holm, et al 2015.

The detection results are shown in fig. 7, and the results show that the serum anti-OVA antibody level of mice in Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptide fragment treatment groups at 300 mu g of immunization dose is not obviously different from that of a control group (PBS) and is obviously lower than that of full-length gp96 immunized mice (all P is less than 0.05), which indicates that polypeptide fragment immunization can not activate effector B cells.

Example 5: application of different truncations based on C-terminal structural domain of gp96 protein in treatment of systemic lupus erythematosus

1. Group immunization of mice

300 female Lyn (-/-) mice with the weight of 14-16 g at six weeks of age were randomly divided into Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, Peptide15 polypeptide, full-length gp96 immune group and control group (PBS) (10 mice each) and treated as follows: when the mice grew to

age

7, 8, 10, 12, 14, and 16 weeks, the purified recombinant polypeptides prepared in examples 1 and 3, full-length gp96 or PBS was subcutaneously injected into the abdomen of the mice at 100, 300, or 500 μ g per mouse, respectively.

When the mice grew to 20 weeks of age, 200. mu.l of blood was collected from the orbit. Standing at room temperature for 30min, centrifuging at 3000rpm for 20min, and collecting supernatant.

2. Analysis of anti-double stranded DNA antibody titer

The level of anti-dsDNA antibodies in serum was determined by enzyme-linked immunosorbent assay (ELISA) as follows:

2.1 to a 96-well microplate (purchased from Nunc, Rochester NY, USA) was added 2.5. mu.g/ml calf thymus dsDNA standard (purchased from Sigma Aldrich, USA, cat # D8899-1MG) at 100. mu.l per well. Coating was carried out overnight at 4 ℃.

2.2 mu.l of 2% bovine serum albumin (from Invitrogen, USA) was added and incubated for 60 min at 37 ℃.

2.3 serum samples (50. mu.l, 1: 50 dilution) and anti-dsDNA antibody standards (purchased from Chemicon International, USA, clone number: 16-13) were added and incubated for 60 min at 37 ℃.

2.4 discard the well liquid, add 200. mu.l of 1 Xwashing solution (0.1% for PBST, PBS added Triton-X100) per well, let stand for 30 seconds, spin-dry, repeat washing 5 times. And finally patting the paper on absorbent paper.

2.5 HRP-labeled anti-mouse secondary antibody (purchased from Chinese fir gold bridge, 1: 5000 dilution) was added in 50. mu.l per well. Incubate at 37 ℃ for 60 minutes.

2.6 discard the liquid in the well, add 200. mu.l of 1 × washing solution into each well, stand for 30 seconds, spin-dry, repeat washing 5 times. And finally patting the paper on absorbent paper.

2.7 mu.l of Substrate (1 XTMB ELISA Substrate Solution, from eBioscience, cat # 00-4201-56) was added per well. Incubate at 37 ℃ for 15 minutes.

2.8 discard the well liquid, 50. mu.l stop buffer (2M H) was added to each well ₂ SO ₄ ) Mixing, and reading at 450nm wavelength on a microplate reader. The measurement should be performed within 15 minutes after the addition of the stop solution.

3. Measurement of urine protein

Urine protein detection kit purchased from Nanjing institute of bioengineering (Cat number: C035-2)

3.1 three tubes were taken and labeled blank, standard and assay. 0.05ml of double distilled water and 3.0ml of CBB reagent are added into the blank tube. The standard tube is added with 563mg/L protein standard solution 0.05ml and CBB reagent 3.0 ml. The assay tube was filled with 0.05ml of urine sample and 3.0ml of CBB reagent.

3.2 mixing well, standing for 5 minutes. The absorbance of each tube was measured with a spectrophotometer at 595 nm.

Urinary protein concentration (mg/L) × (measurement OD value-blank OD value)/(standard OD value-blank OD value) × standard concentration

4. Evaluation of Effect

The results of the detection of the anti-double stranded DNA antibodies in the mice are shown in FIG. 8. The results show that the anti-double-chain DNA antibody titer of the polypeptide-treated mice of Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 is obviously lower than that of a control group (PBS) (P <0.001) and is also obviously lower than that of a full-length gp 96-treated group (P < 0.05). This shows that the effect of the polypeptide for treating systemic lupus erythematosus is better than that of full-length gp 96.

The results of urine protein detection in mice are shown in FIG. 9. The results show that the urinary protein level of Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptide treated mice is significantly lower than that of control group (PBS) (P <0.001) and also significantly lower than that of full-length gp96 treated group (P < 0.05). This indicates that the effect of the polypeptide on treating systemic lupus erythematosus is superior to full-length gp 96.

Example 6: application of different truncations based on C-terminal structural domain of gp96 protein in preventing type 1 diabetes

1. Immunization of mice

300 NOD mice prevention models with the weight of 14-16 g at six weeks of age were randomly divided into Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, Peptide15 polypeptide, full-length gp96 immune group and control group (PBS) (10 mice each) and treated as follows: when the mice grew to

age

7, 8, 10, 12, 14 weeks, the purified recombinant polypeptides prepared in examples 1 and 3, full-length gp96 or PBS was injected subcutaneously into the abdomen of the mice at 100, 300 or 500 μ g per mouse, respectively.

Monitoring blood glucose level of NOD mice from 9 weeks of age, collecting 1 drop of tail venous blood, and measuring with glucometer (ACCU-

Performa) blood glucose values were determined with reference to the instructions and the prevalence was counted. By the blood sugar level>Diabetes mellitus was diagnosed at 13.3mmol/L for 2 or more times (T1D).

2. Evaluation of Effect

The results of the mouse disease index are shown in a graph 10, and the results show that the prevalence rate of Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptide-immunized mice T1D is obviously lower than that of full-length gp 96-immunized mice (P <0.05 or P <0.01), and control mice (PBS) are all attacked at 11-14 weeks.

The blood sugar concentration detection results of the mice are shown in a figure 11, and the results show that the blood sugar concentration of the mice immunized by the Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptides is obviously lower than that of the full-length gp96 immunized group (P < 0.05).

Mice were sacrificed at the age of 20 weeks, spleen cells of the mice were isolated, and the activated CD8 of the mice after immunization was analyzed by flow cytometry ⁺ The percentage of T cells, the assay results are shown in FIG. 12. The results show that the mice in Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptide fragment treated groups are activated CD8 ⁺ The percentage of T cells was significantly lower than that of full-length gp96 group mice (P)<0.05)。

The results show that the prevention effect of the polypeptide on type 1 diabetes is obviously superior to that of full-length gp 96.

Example 7: use of different truncations based on the C-terminal domain of gp96 protein for the treatment of type 1 diabetes

1. Immunization of mice

Treatment models of 100 NOD mice with a weight of 14-16 g at six weeks of age were randomly divided into Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, Peptide15 polypeptide, full-length gp96 immune group and control group (PBS) (10 mice per group), and treated as follows: when the mice grew to

age

14, 15, 17, 19, 21 weeks, the purified polypeptide prepared in examples 1 and 3, full-length gp96 or PBS was injected subcutaneously into the abdomen of the mice at a dose of 300 μ g per mouse, respectively.

Blood glucose levels were monitored from week 1 after the last immunization and tested 1 time per week. Taking 1 drop of tail venous blood of mice, and using a glucometer (ACCU-

Performa) blood glucose values were measured with reference to the instructions.

2. Evaluation of Effect

The results of the blood glucose concentration measurements are shown in FIG. 13. The results show that the blood sugar concentration of mice after being immunized by Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14 and Peptide15 polypeptides is gradually reduced along with the time change, the final concentration is obviously lower than that of a control group (PBS) (P <0.001), and the blood sugar level of mice in all polypeptide immunization groups is also obviously lower than that of a full-length gp96 immunization group (P < 0.05).

The results show that the polypeptide is used for immunizing mice, the TID of the mice can be effectively treated, and the treatment effect of the polypeptide is superior to full-length gp 96.

Example 8: application of mutant polypeptide fragment in treatment of type 1 diabetes

1. Expression of mutant polypeptide fragments

Three amino acid sites randomly selected from the Peptide1 and Peptide15 are mutated into alanine (Ala) which are respectively marked as Peptide 1m-1(SEQ ID NO:55), Peptide 1m-2(SEQ ID NO:56), Peptide15 m-1(SEQ ID NO:59) and Peptide15 m-2(SEQ ID NO:60), and then three amino acid sites in Peptide1 and Peptide15 are deleted and respectively marked as Peptide 1d-1(SEQ ID NO:57), Peptide 1d-2(SEQ ID NO:58), Peptide15d-1(SEQ ID NO:61) and Peptide15d-2(SEQ ID NO: 62). The C-terminal of each protein or polypeptide is added with a 6xHis tag. The recombinant plasmid was synthesized by kasuga biotechnology, ltd, and then expression and purification of the polypeptide were performed as in example 1.

2. Immunization of mice

The treatment models of 60 NOD mice with the weight of 14-16 g at six weeks are randomly divided into Peptide1, Peptide 1m-2, Peptide 1d-1, Peptide 1d-2, Peptide15 m-1, Peptide15 m-2, Peptide15d-1 and Peptide15d-2 polypeptide immune groups (10 in each group), and the following treatment is respectively carried out: when the mice grew to 14, 15, 17, 19 and 21 weeks of age, the polypeptides prepared in example 1 were subcutaneously injected into the abdomen of the mice at a dose of 300. mu.g per mouse.

Performa) blood glucose values were determined with reference to the instructions.

3. Evaluation of Effect

The detection results of the blood glucose concentration of mice immunized with Peptide1 amino acid deletion and substitution mutant polypeptides are shown in fig. 14, and the results show that the blood glucose concentration of the mice immunized with the mutant polypeptides is not obviously different from that of the non-mutant polypeptides, which indicates that the mutant polypeptides and the non-mutant polypeptides have the same treatment function.

The detection results of the blood sugar concentration of mice immunized with Peptide15 amino acid deletion and substitution mutant polypeptides are shown in fig. 15, and the results show that the blood sugar concentration of the mice immunized with the mutant polypeptides is not obviously different from that of the non-mutant polypeptides, which indicates that some mutant polypeptides and non-mutant polypeptides have the same treatment function.

The results show that the polypeptide of the invention can tolerate amino acid mutation to a certain extent and maintain the activity of treating autoimmune diseases.

Example 9: use of different truncations based on the C-terminal domain of the gp96 protein for the treatment of induced mouse arthritic inflammation

1. Group immunization of mice

310 female DBA/1 mice (Beijing Wittison Hua laboratory animal technology Co., Ltd.) with the weight of 14-16 g at six weeks are randomly divided into Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, Peptide15 polypeptide, full-length gp96 immune group, control group (PBS) and model group (10 in each group), and the rest 300 mice are subjected to CIA molding except 10 normal control groups. Dissolving 10mg bovine II type collagen in 5mL of 0.01mmol/L acetic acid solution, shaking overnight at 4 ℃, taking 5mL of complete Freund's adjuvant containing 2mg/mL, mixing in equal volume, emulsifying completely to obtain antigen emulsion, and storing in refrigerator at 4 ℃ for later use. The first immunization is to inject 0.1mL of antigen emulsion into the tail root of the mouse subcutaneously, the booster immunization is carried out once by using 0.1mL of emulsion mixed by II type collagen and incomplete Freund's adjuvant on the 21 st day of model building, and the normal group is injected with physiological saline with the same volume in the same way. The treatment is carried out on the 8 th day after the secondary immunization as follows: mice were grown to

age

10, 11 and 13 weeks, and the recombinant polypeptides prepared in examples 1 and 3, full-length gp96 or PBS was subcutaneously injected into the abdomen of the mice at a dose of 100 μ g, 300 μ g or 500 μ g per mouse, respectively.

Index detection, wherein the arthritis index is given to the joint inflammation index scores on 0, 5, 10, 15, 20, 25 and 30 days after the administration of the mice, and the score standard is as follows: 0 point, no obvious joint red swelling; mild red swelling of ankle joint or toe joint in 1 point; mild red swelling from toe joint to ankle joint in 2 points; 3, mild red swelling from toe joint to ankle joint; the severe redness of the ankle to the entire paw was scored 4 points, with a maximum score of 16 points per mouse. Thickness of sole, the thickness of swelling is measured on the sole with swelling selected on 0, 5, 10, 15, 20, 25 and 30 days after administration of the mouse, and the swelling is measured at the most severe position with a vernier caliper in mm.

2. Evaluation of Effect

The results of joint inflammation indexes of mice are shown in a figure 16, and the results show that the joint inflammation indexes of the mice after polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 8, polypeptide 13, polypeptide 14 and polypeptide 15 polypeptide immunization are obviously lower than those of a model group (P <0.001) and are also lower than those of a full-length gp96 treatment group (P < 0.05). In addition, the results of the sole thickness measurements showed that the sole thickness of mice immunized with peptides Peptide1, Peptide 2, Peptide 3, Peptide 4, Peptide 8, Peptide 13, Peptide 14, and Peptide15 was significantly lower than that of the model group (P <0.001) and also lower than that of the full-length gp96 treatment group (P < 0.05). Similar therapeutic results were obtained with mice injected at doses of 100 μ g or 500 μ g for rheumatoid disease. The results show that the polypeptide is used for immunizing mice, the induced arthritis of the mice can be effectively treated, and the treatment effect is better than full-length gp 96.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Foshan thermal shock Biotechnology Ltd

<120> polypeptide for treating autoimmune disease

<130> IDC200566

<160> 63

<170> PatentIn version 3.5

<210> 1

<211> 2412

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding full-length gp96

<400> 1

atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc tgaccttcgg gtcggtcaga 60

gctgacgatg aagttgatgt ggatggtaca gtagaagagg atctgggtaa aagtagagaa 120

ggatcaagga cggatgatga agtagtacag agagaggaag aagctattca gttggatgga 180

ttaaatgcat cacaaataag agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 240

gaagttaaca gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc 300

ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct aatatcactg 360

actgatgaaa atgctctttc tggaaatgag gaactaacag tcaaaattaa gtgtgataag 420

gagaagaacc tgctgcatgt cacagacacc ggtgtaggaa tgaccagaga agagttggtt 480

aaaaaccttg gtaccatagc caaatctggg acaagcgagt ttttaaacaa aatgactgaa 540

gcacaggaag atggccagtc aacttctgaa ttgattggcc agtttggtgt cggtttctat 600

tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 660

cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 720

ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 780

ttggatacaa ttaaaaatct cgtcaaaaaa tattcacagt tcataaactt tcctatttat 840

gtatggagca gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa 900

gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 960

ccaaagacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 1020

ccaatatggc agagaccatc aaaagaagta gaagaagatg aatacaaagc tttctacaaa 1080

tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1140

gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200

tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260

gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320

gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380

aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440

aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500

tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560

attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620

atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680

aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740

cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1800

agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc tctgctgaat 1860

tggatgaaag ataaagccct taaggacaag attgaaaagg ctgtggtgtc tcagcgcctg 1920

acagaatctc cgtgtgcttt ggtggccagc cagtacggat ggtctggcaa catggagaga 1980

atcatgaaag cacaagcgta ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 2040

cagaagaaaa catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga 2100

attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt gtttgaaaca 2160

gcaacgcttc ggtcagggta tcttttacca gacactaaag catatggaga tagaatagaa 2220

agaatgcttc gcctcagttt gaacattgac cctgatgcaa aggtggaaga agagcccgaa 2280

gaagaacctg aagagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2340

gaaatggatg tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaaaaa 2400

gatgaattgt aa 2412

<210> 2

<211> 93

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding gp96-N

<400> 2

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa taa 93

<210> 3

<211> 498

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding gp96-N + M

<400> 3

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctctaa 498

<210> 4

<211> 414

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding gp96-M

<400> 4

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct ctaa 414

<210> 5

<211> 276

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding gp96-C

<400> 5

atggctgtgg tgctgttcga gaccgccact ctgcgctccg gttatctgct ccccgacacc 60

aaggcctacg gcgatcgtat tgagcgtatg ctgcgtctgt ctctgaatat cgaccccgac 120

gccaaggtgg aggaagaacc cgaggaagaa cccgaggaga ccgctgaaga taccaccgag 180

gacactgaac aagacgaaga cgaggagatg gatgtgggta ccgacgagga ggaagagact 240

gctaaggaat ccaccgccga gaaggacgag ctgtaa 276

<210> 6

<211> 684

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding gp96-C + M

<400> 6

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 420

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 480

gatcgtattg agcgtatgct gcgtctgtct ctgaatatcg accccgacgc caaggtggag 540

gaagaacccg aggaagaacc cgaggagacc gctgaagata ccaccgagga cactgaacaa 600

gacgaagacg aggagatgga tgtgggtacc gacgaggagg aagagactgc taaggaatcc 660

accgccgaga aggacgagct gtaa 684

<210> 7

<211> 803

<212> PRT

<213> Artificial Sequence

<220>

<223> full-length gp96 amino acid sequence

<400> 7

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe

1 5 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu

20 25 30

Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val

35 40 45

Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser

50 55 60

Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala

65 70 75 80

Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn

85 90 95

Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu

100 105 110

Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly

115 120 125

Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu

130 135 140

Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val

145 150 155 160

Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn

165 170 175

Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile

180 185 190

Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys

195 200 205

Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu

210 215 220

Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr

225 230 235 240

Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser

245 250 255

Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser

260 265 270

Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr

275 280 285

Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu

290 295 300

Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys

305 310 315 320

Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met

325 330 335

Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu

340 345 350

Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp

355 360 365

Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys

370 375 380

Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu

385 390 395 400

Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val

405 410 415

Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe

420 425 430

Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg

435 440 445

Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu

450 455 460

Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr

465 470 475 480

Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val

485 490 495

Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe

500 505 510

Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val

515 520 525

Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser

530 535 540

Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys

545 550 555 560

Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys

565 570 575

Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

580 585 590

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

595 600 605

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

610 615 620

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

625 630 635 640

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

645 650 655

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

660 665 670

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

675 680 685

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

690 695 700

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

705 710 715 720

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

725 730 735

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

740 745 750

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

755 760 765

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

770 775 780

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys

785 790 795 800

Asp Glu Leu

<210> 8

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> gp96-N amino acid sequence

<400> 8

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln

20 25 30

<210> 9

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223> gp96-N + M amino acid sequence

<400> 9

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu

165

<210> 10

<211> 137

<212> PRT

<213> Artificial Sequence

<220>

<223> gp96-M amino acid sequence

<400> 10

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu

130 135

<210> 11

<211> 91

<212> PRT

<213> Artificial Sequence

<220>

<223> gp96-C amino acid sequence

<400> 11

Met Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu

1 5 10 15

Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg

20 25 30

Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu

35 40 45

Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln

50 55 60

Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr

65 70 75 80

Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

85 90

<210> 12

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> gp96-C + M amino acid sequence

<400> 12

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

130 135 140

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

145 150 155 160

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

165 170 175

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

180 185 190

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

195 200 205

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys

210 215 220

Asp Glu Leu

225

<210> 13

<211> 768

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide1

<400> 13

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctcgctgt ggtgctgttc gagaccgcca ctctgcgctc cggttatctg 540

ctccccgaca ccaaggccta cggcgatcgt attgagcgta tgctgcgtct gtctctgaat 600

atcgaccccg acgccaaggt ggaggaagaa cccgaggaag aacccgagga gaccgctgaa 660

gataccaccg aggacactga acaagacgaa gacgaggaga tggatgtggg taccgacgag 720

gaggaagaga ctgctaagga atccaccgcc gagaaggacg agctgtaa 768

<210> 14

<211> 678

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 2

<400> 14

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctcgctgt ggtgctgttc gagaccgcca ctctgcgctc cggttatctg 540

ctccccgaca ccaaggccta cggcgatcgt attgagcgta tgctgcgtct gtctctgaat 600

atcgaccccg acgccaaggt ggaggaagaa cccgaggaag aacccgagga gaccgctgaa 660

gataccaccg aggactaa 678

<210> 15

<211> 588

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 3

<400> 15

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctcgctgt ggtgctgttc gagaccgcca ctctgcgctc cggttatctg 540

ctccccgaca ccaaggccta cggcgatcgt attgagcgta tgctgtaa 588

<210> 16

<211> 498

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 4

<400> 16

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctctaa 498

<210> 17

<211> 408

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 5

<400> 17

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagtaa 408

<210> 18

<211> 318

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 6

<400> 18

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttaa 318

<210> 19

<211> 228

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 7

<400> 19

atggaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 60

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 120

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 180

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagctaa 228

<210> 20

<211> 684

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 8

<400> 20

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 420

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 480

gatcgtattg agcgtatgct gcgtctgtct ctgaatatcg accccgacgc caaggtggag 540

gaagaacccg aggaagaacc cgaggagacc gctgaagata ccaccgagga cactgaacaa 600

gacgaagacg aggagatgga tgtgggtacc gacgaggagg aagagactgc taaggaatcc 660

accgccgaga aggacgagct gtaa 684

<210> 21

<211> 594

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 9

<400> 21

atgcgcgaag ccgtggagaa agagtttgag cctctgctca actggatgaa agacaaggcc 60

ctcaaagaca agatcgaaaa ggccgtggtg tcccagcgtc tcactgaaag cccttgcgct 120

ctggtggcca gccagtacgg ttggagcggc aatatggagc gtatcatgaa ggctcaagct 180

taccaaaccg gcaaggacat ctccaccaac tactacgcct cccaaaagaa gaccttcgag 240

atcaaccctc gccatcctct gatccgcgat atgctgcgcc gtatcaagga agatgaggac 300

gacaagaccg tgctggacct cgctgtggtg ctgttcgaga ccgccactct gcgctccggt 360

tatctgctcc ccgacaccaa ggcctacggc gatcgtattg agcgtatgct gcgtctgtct 420

ctgaatatcg accccgacgc caaggtggag gaagaacccg aggaagaacc cgaggagacc 480

gctgaagata ccaccgagga cactgaacaa gacgaagacg aggagatgga tgtgggtacc 540

gacgaggagg aagagactgc taaggaatcc accgccgaga aggacgagct gtaa 594

<210> 22

<211> 504

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 10

<400> 22

atgcagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 60

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 120

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 180

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 240

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 300

gatcgtattg agcgtatgct gcgtctgtct ctgaatatcg accccgacgc caaggtggag 360

gaagaacccg aggaagaacc cgaggagacc gctgaagata ccaccgagga cactgaacaa 420

gacgaagacg aggagatgga tgtgggtacc gacgaggagg aagagactgc taaggaatcc 480

accgccgaga aggacgagct gtaa 504

<210> 23

<211> 414

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 11

<400> 23

atgcaaaccg gcaaggacat ctccaccaac tactacgcct cccaaaagaa gaccttcgag 60

atcaaccctc gccatcctct gatccgcgat atgctgcgcc gtatcaagga agatgaggac 120

gacaagaccg tgctggacct cgctgtggtg ctgttcgaga ccgccactct gcgctccggt 180

tatctgctcc ccgacaccaa ggcctacggc gatcgtattg agcgtatgct gcgtctgtct 240

ctgaatatcg accccgacgc caaggtggag gaagaacccg aggaagaacc cgaggagacc 300

gctgaagata ccaccgagga cactgaacaa gacgaagacg aggagatgga tgtgggtacc 360

gacgaggagg aagagactgc taaggaatcc accgccgaga aggacgagct gtaa 414

<210> 24

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 12

<400> 24

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 60

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 120

gatcgtattg agcgtatgct gcgtctgtct ctgaatatcg accccgacgc caaggtggag 180

gaagaacccg aggaagaacc cgaggagacc gctgaagata ccaccgagga cactgaacaa 240

gacgaagacg aggagatgga tgtgggtacc gacgaggagg aagagactgc taaggaatcc 300

accgccgaga aggacgagct gtaa 324

<210> 25

<211> 594

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 13

<400> 25

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 420

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 480

gatcgtattg agcgtatgct gcgtctgtct ctgaatatcg accccgacgc caaggtggag 540

gaagaacccg aggaagaacc cgaggagacc gctgaagata ccaccgagga ctaa 594

<210> 26

<211> 504

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 14

<400> 26

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct cgctgtggtg 420

ctgttcgaga ccgccactct gcgctccggt tatctgctcc ccgacaccaa ggcctacggc 480

gatcgtattg agcgtatgct gtaa 504

<210> 27

<211> 414

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide15

<400> 27

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgag atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct ctaa 414

<210> 28

<211> 324

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 16

<400> 28

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gtaa 324

<210> 29

<211> 234

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 17

<400> 29

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttaa 234

<210> 30

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide1 amino acid sequence

<400> 30

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

165 170 175

Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu

180 185 190

Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu

195 200 205

Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu

210 215 220

Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu

225 230 235 240

Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

245 250 255

<210> 31

<211> 225

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 2 amino acid sequence

<400> 31

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

165 170 175

Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu

180 185 190

Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu

195 200 205

Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu

210 215 220

Asp

225

<210> 32

<211> 195

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 3 amino acid sequence

<400> 32

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

165 170 175

Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu

180 185 190

Arg Met Leu

195

<210> 33

<211> 165

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 4 amino acid sequence

<400> 33

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu

165

<210> 34

<211> 135

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 5 amino acid sequence

<400> 34

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys

130 135

<210> 35

<211> 105

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 6 amino acid sequence

<400> 35

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly

100 105

<210> 36

<211> 75

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 7 amino acid sequence

<400> 36

Met Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu Pro Glu Phe Asp

1 5 10 15

Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val Lys Phe Asp Glu

20 25 30

Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu Lys Glu Phe Glu

35 40 45

Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys Asp Lys Ile Glu

50 55 60

Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser

65 70 75

<210> 37

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 8 amino acid sequence

<400> 37

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

130 135 140

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

145 150 155 160

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

165 170 175

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

180 185 190

Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val

195 200 205

Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys

210 215 220

Asp Glu Leu

225

<210> 38

<211> 197

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 9 amino acid sequence

<400> 38

Met Arg Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met

1 5 10 15

Lys Asp Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln

20 25 30

Arg Leu Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp

35 40 45

Ser Gly Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly

50 55 60

Lys Asp Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu

65 70 75 80

Ile Asn Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys

85 90 95

Glu Asp Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe

100 105 110

Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala

115 120 125

Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp

130 135 140

Pro Asp Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr

145 150 155 160

Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met

165 170 175

Asp Val Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala

180 185 190

Glu Lys Asp Glu Leu

195

<210> 39

<211> 167

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 10 amino acid sequence

<400> 39

Met Gln Arg Leu Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr

1 5 10 15

Gly Trp Ser Gly Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln

20 25 30

Thr Gly Lys Asp Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr

35 40 45

Phe Glu Ile Asn Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg

50 55 60

Ile Lys Glu Asp Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val

65 70 75 80

Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr

85 90 95

Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn

100 105 110

Ile Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu

115 120 125

Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu

130 135 140

Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser

145 150 155 160

Thr Ala Glu Lys Asp Glu Leu

165

<210> 40

<211> 137

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 11 amino acid sequence

<400> 40

Met Gln Thr Gly Lys Asp Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys

1 5 10 15

Lys Thr Phe Glu Ile Asn Pro Arg His Pro Leu Ile Arg Asp Met Leu

20 25 30

Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys Thr Val Leu Asp Leu Ala

35 40 45

Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro

50 55 60

Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg Leu Ser

65 70 75 80

Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu Glu Glu

85 90 95

Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu

100 105 110

Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys

115 120 125

Glu Ser Thr Ala Glu Lys Asp Glu Leu

130 135

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 12 amino acid sequence

<400> 41

Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys Thr Val Leu Asp

1 5 10 15

Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly Tyr Leu

20 25 30

Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met Leu Arg

35 40 45

Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu Pro Glu

50 55 60

Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr Glu Gln

65 70 75 80

Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu Thr

85 90 95

Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

100 105

<210> 42

<211> 197

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 13 amino acid sequence

<400> 42

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

130 135 140

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

145 150 155 160

Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp

165 170 175

Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu

180 185 190

Asp Thr Thr Glu Asp

195

<210> 43

<211> 167

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 14 amino acid sequence

<400> 43

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr

130 135 140

Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly

145 150 155 160

Asp Arg Ile Glu Arg Met Leu

165

<210> 44

<211> 137

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide15 amino acid sequence

<400> 44

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu

130 135

<210> 45

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 16 amino acid sequence

<400> 45

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys

100 105

<210> 46

<211> 77

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide 17 amino acid sequence

<400> 46

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly

65 70 75

<210> 47

<211> 768

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 1m-1

<400> 47

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagcgcg 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcaac 420

cctcgccatc ctctgatccg cgatatggca cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctcgctgt ggtgctgttc gagaccgcca ctctgcgctc cggttatctg 540

ctccccgaca ccaaggccta cggcgatcgt attgagcgta tgctgcgtct gtctctgaat 600

atcgaccccg acgccaaggt ggaggaagaa cccgaggaag aacccgagga gaccgctgaa 660

gataccaccg aggacactga acaagacgaa gacgaggaga tggatgtggg taccgacgca 720

gaggaagaga ctgctaagga atccaccgcc gagaaggacg agctgtaa 768

<210> 48

<211> 768

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 1m-2

<400> 48

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatcgcc gctctccccg agttcgatgg caagcgcttc 120

cagaacgtgg ccaaggaggg cgtcaaattc gatgagagcg agaagaccaa ggagagccgc 180

gaagccgtgg agaaagagtt tgagcctctg ctcaactgga tgaaagacaa ggccctcaaa 240

gacaagatcg aaaaggccgt ggtgtcccag cgtctcactg aaagcccttg cgctctggtg 300

gccagccagt acggttggag cggcaatatg gagcgtatca tgaaggctca agcttaccaa 360

accggcaagg acatctccac caactactac gcctcccaaa agaagacctt cgagatcgcg 420

cctcgccatc ctctgatccg cgatatgctg cgccgtatca aggaagatga ggacgacaag 480

accgtgctgg acctcgctgt ggtgctgttc gagaccgcca ctctgcgctc cggttatctg 540

ctccccgaca ccaaggccta cggcgatcgt attgagcgta tgctgcgtct gtctctgaat 600

atcgaccccg acgccaaggt ggaggaagaa cccgaggaag aacccgagga gaccgctgaa 660

gataccaccg aggacactga acaagacgaa gacgaggcga tggatgtggg taccgacgag 720

gaggaagaga ctgctaagga atccaccgcc gagaaggacg agctgtaa 768

<210> 49

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 1d-1

<400> 49

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatcgct ctccccgagt tcgatggcaa gcgcttccag 120

aacgtggcca aggagggcgt caaattcgat gagagcgaga agaccaagga gagccgcgaa 180

gccgtggaga aagagtttga gcctctgctc aactggatga aagacaaggc cctcaaagac 240

aagatcgaaa aggccgtggt gtcccagcgt ctcactgaaa gcccttgcgc tctggtggcc 300

agccagtacg gttggagcgg caatatggag cgtatcatga aggctcaagc ttaccaaacc 360

ggcaaggaca tctccaccaa ctactacgcc tcccaaaaga agaccttcga gatccctcgc 420

catcctctga tccgcgatat gctgcgccgt atcaaggaag atgaggacga caagaccgtg 480

ctggacctcg ctgtggtgct gttcgagacc gccactctgc gctccggtta tctgctcccc 540

gacaccaagg cctacggcga tcgtattgag cgtatgctgc gtctgtctct gaatatcgac 600

cccgacgcca aggtggagga agaacccgag gaagaacccg aggagaccgc tgaagatacc 660

accgaggaca ctgaacaaga cgacgaggag atggatgtgg gtaccgacga ggaggaagag 720

actgctaagg aatccaccgc cgagaaggac gagctgtaa 759

<210> 50

<211> 759

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide 1d-2

<400> 50

atggaatcct cccccttcgt ggagcgtctg ctcaagaagg gctacgaagt gatctatctg 60

accgaacccg tggacgagta ttgcatccaa gctctccccg agttcgatgg caagcgccag 120

aacgtggcca aggagggcgt caaattcgat gagagcgaga agaccaagga gagccgcgaa 180

gccgtggaga aagagtttga gcctctgctc aactggatga aagacaaggc cctcaaagac 240

aagatcgaaa aggccgtggt gtcccgtctc actgaaagcc cttgcgctct ggtggccagc 300

cagtacggtt ggagcggcaa tatggagcgt atcatgaagg ctcaagctta ccaaaccggc 360

aaggacatct ccaccaacta ctacgcctcc caaaagaaga ccttcgagat caaccctcgc 420

catcctctga tccgcgatat gctgcgccgt atcaaggaag atgaggacga caagaccgtg 480

ctggacctcg ctgtggtgct gttcgagacc gccactctgc gctccggtta tctgctcccc 540

gacaccaagg cctacggcga tcgtattgag cgtatgctgc gtctgtctct gaatatcgac 600

cccgacgcca aggtggagga agaacccgag gaagaacccg aggagaccgc tgaagatacc 660

accgaggaca ctgaacaaga cgaagacgag gagatggatg tgggtaccga cgaggaagag 720

actgctaagg aatccaccgc cgagaaggac gagctgtaa 759

<210> 51

<211> 414

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide15 m-1

<400> 51

atgcaagctc tccccgagtt cgatggcgca cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggcg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgcg atcaaccctc gccatcctct gatccgcgat 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct ctaa 414

<210> 52

<211> 414

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide15 m-2

<400> 52

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgcg 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttgag 120

cctctgctca actggatgaa agacaaggcc ctcaaagaca agatcgaaaa ggccgtggtg 180

tcccagcgtc tcactgaaag cccttgcgct ctggtggcca gccagtacgg ttggagcggc 240

aatatggagc gtatcatgaa ggctcaagct taccaaaccg gcaaggacat ctccaccaac 300

tactacgcct cccaaaagaa gaccttcgcg atcaaccctc gccatcctct gatccgcgca 360

atgctgcgcc gtatcaagga agatgaggac gacaagaccg tgctggacct ctaa 414

<210> 53

<211> 405

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide15d-1

<400> 53

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaagagc cgcgaagccg tggagaaaga gtttgagcct 120

ctgctcaact ggatgaaaga caaggccctc aaagacaaga tcgaaaaggc cgtggtgtcc 180

cagcgtctca ctgaaagccc ttgcgctctg gtggccagcc agtacggttg gagcggcaat 240

atggagcgta tcatgaaggc tcaataccaa accggcaagg acatctccac caactactac 300

gcctcccaaa agaagacctt cgagatcaac cctcgccatc ctctgatccg cgatatgctg 360

cgccgtatca aggaagatga ggacaagacc gtgctggacc tctaa 405

<210> 54

<211> 405

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence encoding Peptide15d-2

<400> 54

atgcaagctc tccccgagtt cgatggcaag cgcttccaga acgtggccaa ggagggcgtc 60

aaattcgatg agagcgagaa gaccaaggag agccgcgaag ccgtggagaa agagtttcct 120

ctgctcaact ggatgaaaga caaggccctc aaagacaaga tcgaaaaggc cgtggtgtcc 180

cagcgtctca ctgaaagccc ttgcgctctg gtggccagcc agtacggttg gagcggcaat 240

atggagcgta tcatgaaggc tcaagcttac caaaccggca aggacatctc caccaactac 300

tacgcctccc aaaagaagac cttcgagatc aaccctcgcc atcctctgat ccgcatgctg 360

cgccgtatca aggaagatga ggacaagacc gtgctggacc tctaa 405

<210> 55

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> 1m-1 amino acid sequence

<400> 55

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Ala Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Ala Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

165 170 175

Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu

180 185 190

Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu

195 200 205

Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu

210 215 220

Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Ala

225 230 235 240

Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

245 250 255

<210> 56

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> 1m-2 amino acid sequence

<400> 56

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Ala Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val

35 40 45

Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu

50 55 60

Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys

65 70 75 80

Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro

85 90 95

Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg

100 105 110

Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn

115 120 125

Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Ala Pro Arg His Pro

130 135 140

Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys

145 150 155 160

Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg

165 170 175

Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu

180 185 190

Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu

195 200 205

Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu

210 215 220

Asp Thr Glu Gln Asp Glu Asp Glu Ala Met Asp Val Gly Thr Asp Glu

225 230 235 240

Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

245 250 255

<210> 57

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> 1d-1 amino acid sequence

<400> 57

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Ala Leu Pro

20 25 30

Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala Lys Glu Gly Val Lys

35 40 45

Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu Lys

50 55 60

Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys Asp

65 70 75 80

Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr Glu Ser Pro Cys

85 90 95

Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg Ile

100 105 110

Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn Tyr

115 120 125

Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Pro Arg His Pro Leu Ile

130 135 140

Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys Thr Val

145 150 155 160

Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly

165 170 175

Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met

180 185 190

Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu

195 200 205

Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr

210 215 220

Glu Gln Asp Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu Glu

225 230 235 240

Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

245 250

<210> 58

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> 1d-2 amino acid sequence

<400> 58

Met Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu

1 5 10 15

Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu

20 25 30

Pro Glu Phe Asp Gly Lys Arg Gln Asn Val Ala Lys Glu Gly Val Lys

35 40 45

Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu Lys

50 55 60

Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys Asp

65 70 75 80

Lys Ile Glu Lys Ala Val Val Ser Arg Leu Thr Glu Ser Pro Cys Ala

85 90 95

Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn Met Glu Arg Ile Met

100 105 110

Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp Ile Ser Thr Asn Tyr Tyr

115 120 125

Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg His Pro Leu Ile

130 135 140

Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp Asp Lys Thr Val

145 150 155 160

Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser Gly

165 170 175

Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg Met

180 185 190

Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu Glu

195 200 205

Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp Thr

210 215 220

Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu Glu

225 230 235 240

Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Leu

245 250

<210> 59

<211> 137

<212> PRT

<213> Artificial Sequence

<220>

<223> 15m-1 amino acid sequence

<400> 59

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Ala Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Ala Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Ala

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu

130 135

<210> 60

<211> 137

<212> PRT

<213> Artificial Sequence

<220>

<223> 15m-2 amino acid sequence

<400> 60

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Ala Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp

35 40 45

Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu

50 55 60

Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly

65 70 75 80

Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp

85 90 95

Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Ala

100 105 110

Pro Arg His Pro Leu Ile Arg Asp Met Ala Arg Arg Ile Lys Glu Asp

115 120 125

Glu Asp Asp Lys Thr Val Leu Asp Leu

130 135

<210> 61

<211> 134

<212> PRT

<213> Artificial Sequence

<220>

<223> 15d-1 amino acid sequence

<400> 61

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Glu

20 25 30

Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys

35 40 45

Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr

50 55 60

Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn

65 70 75 80

Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Thr Gly Lys Asp Ile Ser

85 90 95

Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg

100 105 110

His Pro Leu Ile Arg Asp Met Arg Arg Ile Lys Glu Asp Glu Asp Asp

115 120 125

Lys Thr Val Leu Asp Leu

130

<210> 62

<211> 134

<212> PRT

<213> Artificial Sequence

<220>

<223> 15d-2 amino acid sequence

<400> 62

Met Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala

1 5 10 15

Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg

20 25 30

Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Asn Trp Met Lys Asp Lys

35 40 45

Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu Thr

50 55 60

Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly Asn

65 70 75 80

Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Thr Gly Lys Asp Ile Ser

85 90 95

Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn Pro Arg

100 105 110

His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp Glu Asp

115 120 125

Asp Thr Val Leu Asp Leu

130

<210> 63

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> 6XHis tag nucleotide sequence

<400> 63

caccaccacc atcaccac

18

Claims

1. An isolated polypeptide or variant thereof, wherein the polypeptide consists of at least 136 contiguous amino acid residues of gp96 protein and comprises: amino acid residues 578-713 of the gp96 protein;

wherein the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1 or a few (e.g., 1, 2, 3, 4 or 5) amino acid residues and retains the biological function of the polypeptide from which it is derived (e.g., induces regulatory T cell activation, does not induce effector T cell and B cell activation, prevents and/or treats autoimmune diseases, reduces the level of anti-double stranded DNA antibodies, reduces the level of urine protein, and/or reduces blood glucose).

2. The isolated polypeptide or variant thereof of claim 1, wherein the isolated polypeptide comprises: the protein gp96 comprises the amino acid residues at 578-position 713, 578-position 743, 578-position 773, 578-position 803, 550-position 713, 550-position 743, 550-position 773 or 550-position 803.

3. The isolated polypeptide or variant thereof of claim 1 or 2, wherein the gp96 protein has a sequence as set forth in SEQ ID NO. 7.

4. The isolated polypeptide or variant thereof of any one of claims 1-3, wherein the isolated polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs 30, 31, 32, 33, 37, 42, 43, 44.

5. The isolated polypeptide or variant thereof of any one of claims 1-4, wherein the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1, 2 or 3 amino acid residues;

preferably, the substitution comprises replacing the amino acid residue with alanine (a).

6. The isolated polypeptide or variant thereof of any one of claims 1-5, wherein the variant comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 55-62.

7. A fusion protein comprising the isolated polypeptide or variant thereof of any one of claims 1-6 and an additional polypeptide;

preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the polypeptide or variant thereof, optionally via a linker;

preferably, the additional polypeptide is selected from a protein tag, a targeting moiety, or any combination thereof.

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated polypeptide or variant thereof of any one of claims 1-6, or the fusion protein of claim 7;

preferably, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (i) a sequence shown as any one of SEQ ID NOs 13-16, 20, 25-27 and 47-54; (ii) (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity compared to the sequence of (i); (iii) (iii) a sequence that hybridizes under stringent conditions to the sequence described in (i) or (ii); or (iv) the complement of the sequence described in (i) or (ii).

9. A vector comprising the isolated nucleic acid molecule of claim 8.

10. A host cell comprising the isolated nucleic acid molecule of claim 8 or the vector of claim 9.

11. A pharmaceutical composition comprising the isolated polypeptide or variant thereof of any one of claims 1-6, the fusion protein of claim 7, the isolated nucleic acid molecule of claim 8, the vector of claim 9, or the host cell of claim 10, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition comprises one or more of the isolated polypeptide or variant thereof of any one of claims 1-6, the fusion protein of claim 7.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent;

preferably, the additional pharmaceutically active agent is a drug having activity in the treatment of autoimmune diseases, such as an anti-inflammatory drug or an immunosuppressant.

13. Use of the isolated polypeptide or variant thereof of any one of claims 1-6, the fusion protein of claim 7, the isolated nucleic acid molecule of claim 8, the vector of claim 9 or the host cell of claim 10, or the pharmaceutical composition of claim 11 or 12 in the manufacture of a medicament for:

(i) preventing and/or treating an autoimmune disease in a subject;

(ii) reducing the level of anti-double stranded DNA antibodies in the subject;

(iii) reducing the level of urinary protein in the subject; and/or

(iv) Lowering blood glucose in a subject.

Use of the C-terminal domain of gp96 protein, or an active fragment or variant thereof, in the manufacture of a medicament for:

(i) preventing and/or treating an autoimmune disease in a subject;

(ii) reducing the level of anti-double stranded DNA antibodies in the subject;

(iii) reducing the level of urinary protein in the subject; and/or

(iv) Lowering blood glucose in a subject.

15. The use according to claim 14, wherein the active fragment comprises amino acid residues 578-713 of the gp96 protein;

preferably, the active fragment comprises: the amino acid residues at the 578-713 st position, the 578-743 st position, the 578-773 rd position, the 578-803 th position, the 550-713 st position, the 550-743 th position or the 550-773 th position of the gp96 protein;

preferably, the active fragment comprises an amino acid sequence selected from the group consisting of: 31, 32, 33, 37, 42, 43, 44.

16. The use of claim 14 or 15, wherein the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1 or a few (e.g., 1, 2, 3, 4 or 5) amino acid residues and retains the biological function of the polypeptide from which it is derived (e.g., induces regulatory T cell activation, does not induce activity of effector T and B cell activation, prevents and/or treats autoimmune diseases, reduces the level of anti-double stranded DNA antibodies, reduces the level of urine protein, and/or reduces blood glucose);

preferably, the variant differs from the polypeptide from which it is derived only by substitution, deletion or addition of 1, 2 or 3 amino acid residues; preferably, the substitution comprises replacing the amino acid residue with alanine (a);

preferably, the variant comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 55-62.

17. The use of any one of claims 13-16, wherein the autoimmune disease is selected from systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, ulcerative colitis, crohn's disease, myasthenia gravis, or polymyositis.

18. The use of any one of claims 13-17, wherein the subject is a mammal, such as a human or a mouse.